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DRUG D I SCOVERY
Repurposing of the antihistamine chlorcyclizine and relatedcompounds for treatment of hepatitis C virus infectionShanshan He,1 Billy Lin,1 Virginia Chu,1 Zongyi Hu,1 Xin Hu,2 Jingbo Xiao,2 Amy Q. Wang,2
Cameron J. Schweitzer,1 Qisheng Li,1 Michio Imamura,3 Nobuhiko Hiraga,3 Noel Southall,2
Marc Ferrer,2 Wei Zheng,2 Kazuaki Chayama,3 Juan J. Marugan,2 T. Jake Liang1*
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Hepatitis Cvirus (HCV) infectionaffects anestimated185millionpeopleworldwide,with chronic infectionoften leading toliver cirrhosis and hepatocellular carcinoma. Although HCV is curable, there is an unmet need for the development ofeffective and affordable treatment options. Througha cell-basedhigh-throughput screen,we identified chlorcyclizineHCl(CCZ), an over-the-counter drug for allergy symptoms, as a potent inhibitor of HCV infection. CCZ inhibited HCV infectionin human hepatoma cells and primary human hepatocytes. Themode of action of CCZ ismediated by inhibiting an earlystageofHCV infection, probably targeting viral entry intohost cells. The in vitro antiviral effect of CCZwas synergisticwithother anti-HCV drugs, including ribavirin, interferon-a, telaprevir, boceprevir, sofosbuvir, daclatasvir, and cyclosporin A,without significant cytotoxicity, suggesting its potential in combination therapy of hepatitis C. In the mouse pharmaco-kinetic model, CCZ showed preferential liver distribution. In chimeric mice engrafted with primary human hepatocytes,CCZ significantly inhibited infection of HCV genotypes 1b and 2a, without evidence of emergence of drug resistance,during 4 and 6 weeks of treatment, respectively. With its established clinical safety profile as an allergy medication, af-fordability, and a simple chemical structure for optimization, CCZ represents a promising candidate for drug repurposingand further development as an effective and accessible agent for treatment of HCV infection.
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INTRODUCTION
Hepatitis C virus (HCV) chronic infection is usually asymptomaticand many individuals are unaware of their infection. However, with-out treatment, HCV infection hastens the development of liver dis-eases, including cirrhosis, liver failure, and hepatocellular carcinoma(1). In fact, more than 50% of incident hepatocellular carcinoma isdue to HCV infection, which is the fastest-growing cause of cancer-related death in the United States (2, 3). In 2012, the U.S. Centers forDisease Control and Prevention recommended screening for HCV in-fection among all persons born between 1945 and 1965 (4). It is an-ticipated that a large number of infected individuals would be identifiedthrough such a screening effort. Although HCV is spread worldwide,it is particularly prevalent in Asia and Africa, as well as in high-risk pop-ulations such as intravenous drug users. With hundreds of millions ofthe population affected by chronic hepatitis C infection, it is essentialthat effective and affordable drugs are developed or repurposed totreat the chronic infection.
A protective vaccine for HCV is not yet available (5), and pegylatedinterferon (PEG-IFN) and ribavirin (RBV) have been the cornerstoneof HCV therapy for many years. Several direct-acting antivirals have beenapproved by the U.S. Food and Drug Administration (FDA) for triple-therapy regimens in combination with PEG-IFN and RBV, leading toimprovement of viral clearance rate in genotype 1–infected patients (1).However, direct-acting antivirals that target viral factors are costly andhave a low genetic barrier to resistance, side effects, and potential fordrug-drug interaction. Recently, several all-oral, interferon (IFN)–freeregimens were introduced for the treatment of chronic HCV infection.These regimens, although very effective, are associated with high costs
1Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases,National Institutes of Health, Bethesda, MD 20892, USA. 2National Center for AdvancingTranslational Sciences, National Institutes of Health, Rockville, MD 20850, USA. 3Depart-ment of Medicine and Molecular Sciences, Graduate School of Biomedical Sciences,Hiroshima University, Hiroshima 730-0053, Japan.*Corresponding author. E-mail: [email protected]
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(for example, a 12-week Sovaldi treatment costs about $84,000), whichgenerally precludes treating the populations most affected by HCV (6).There remains a great need to improve the use of existing drugs and todevelop new drugs and therapeutic targets for HCV therapy to achievethe following features: activity against all genotypes, a high genetic bar-rier to drug resistance, a good safety profile, oral delivery, and globalaffordability.
Existing pharmacopeia can be repurposed to achieve unmet ther-apeutic needs (7). Here, we sought to screen existing, FDA-approveddrugs against HCV infection. Using our previously developed cell-basedquantitative high-throughput screening (qHTS) platform (8), we screeneda comprehensive library of approved drugs that was built by the Na-tional Institutes of Health (NIH) Chemical Genomics Center (NCGC),named the NCGC Pharmaceutical Collection (NPC) (9), to search forapproved drugs with novel anti-HCV activity and potentially newtherapeutic targets. We identified multiple H1-antihistamines with anti-HCV activity. Among these, chlorcyclizine HCl (CCZ), a first-generation an-tihistamine approved in the 1940s, showed high antiviral activity thatwas synergistic with various approved anti-HCV drugs in vitro. Fur-ther in vivo studies in primary human hepatocyte–engrafted miceconfirmed its efficacy in restricting genotypes 1b and 2a HCV infec-tion. The findings from this study, together with the established safetyprofile of CCZ in patients, affordability, and simple chemical structureamenable for further optimization, make CCZ a promising anti-HCVcandidate for further investigation, optimization, and repurposing foruse in HCV-endemic regions and populations.
RESULTS
Identification and confirmation of CCZ analogs asanti-HCV agents from high-throughput screeningA cell-based qHTS of the NPC was carried out against HCV genotype2a (JFH-1 strain) using the platform described previously (8). The
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anti-HCV activity and selectivity were further evaluated using a dose-response study with HCV-Luc (infectious HCV harboring a Renillaluciferase reporter gene) and ATPlite assays (cytotoxicity). The com-pound concentration that led to 50% inhibition and cytotoxicity (EC50
and CC50, respectively) was determined. From about 3800 small-molecule entities in the collection, 118 primary hits were identifiedon the basis of the curve classes of dose-response curves from theluciferase and ATPlite assays (curve classes defined in Materials andMethods) (8) (Fig. 1A). Out of 62 H1-antihistamines with diverse struc-tural features in the NPC, compounds with the cyclizine moiety demon-strated potent activities in the primary screen and single-dose confirmationassay (table S1). Histamine was also included in table S1 as a controlbecause it showed no anti-HCV activity.
In a secondary confirmation assay, CCZ, homochlorcyclizine, andhydroxyzine demonstrated low EC50 values (~50 nM), with high se-lective indices (SI = CC50/EC50) ranging from 318 to 924 (Fig. 1, B andC). Cyclizine lacking a chlorine substitution exhibited a 10-fold loweractivity than CCZ. Cetirizine was reported as a metabolite of hydrox-yzine, but it was not active in inhibiting HCV infection. However,cetirizine amide still showed good activity against HCV (EC50 = 0.103 mM)(Fig. 1C). The low activity of cetirizine could be attributed to its highpolarity and low permeability into cells.
The analogs discussed above—CCZ, homochlorcyclizine, hydroxy-zine, cetirizine, and cetirizine amide—are racemic mixtures of (R)- and
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(S)-enantiomers. To identify whether the configuration affects the anti-HCV activities, we evaluated the (R)- and (S)-enantiomers of CCZ, andthey exhibited no significant difference in EC50 and CC50 values onHCV infection in human hepatoma cells (Huh7.5.1 cell line) (Fig. 1,B and C, and Table 1). The primary metabolite of CCZ or cyclizinein vivo is nor-chlorcyclizine (nor-CCZ) or nor-cyclizine (Fig. 1B),which has little antihistamine activity (10, 11). The antiviral activityof nor-CCZ was comparable to that of CCZ, but with higher cyto-toxicity (Fig. 1C). Under the condition of our cell-based assay, a neg-ligible amount of CCZ was transformed to nor-CCZ (table S2).
Anti-HCV activities of CCZRacemic (R)- and (S)-CCZ were further evaluated against wild-typecell culture–derived HCV (HCVcc; genotype 2a, JFH-1 strain) infec-tion in Huh7.5.1 cells. Intracellular and extracellular viral RNA levelswere significantly reduced with the treatment of racemic, (R)-, and(S)-CCZ compared with DMSO treatment (Fig. 2A). These results fur-ther confirmed that the anti-HCV activity of CCZ analogs is indepen-dent of their configurations. However, (S)-CCZ did show less histaminereceptor inhibitory effect than (R)-CCZ (Table 1); (S)-CCZ was thuschosen for further evaluation.
(S)-CCZ inhibited intracellular HCV RNA level against wild-typeHCVcc infection in primary human hepatocytes in a dose-dependentmanner (Fig. 2B). When Huh7.5.1 cells were infected with HCV chimeric
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Fig. 1. Activities of NPC compounds in qHTS and confirmation of CCZanalogs. (A) Three-axis plot of the activities of NPC compounds in the qHTS.
in curve class 4. (B and C) Chemical structures (B) and in vitro dose-responsecurves (C) of CCZ analogs identified in the NPC screen. The percent inhibition of
Activity (%) was calculated by normalizing luciferase signals to the mean signalfrom the dimethyl sulfoxide (DMSO) control wells. Compounds were sortedaccording to curve classes. Red, active compounds in curve classes 1 and 2;green, weakly active compounds in curve class 3; blue, inactive compounds
the compound in the HCV-Luc assay is shown in blue triangles, and the cyto-toxicity effect in the ATPlite assay of host cells is shown in red circles. EC50 andCC50 values are indicated. Data are means ± SEM (n ≥ 3 replicates). Curves arerepresentative results from at least three independent experiments.
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Fig. 2. Anti-HCV activities of CCZ. (A) Human hepatocytes (Huh7.5.1 genotype 1a, and replicon genotype 1b and 2a assays. Bafilomycin A1
cells) were infected with wild-type HCVcc in the presence of the com-pounds at 10 mM overnight followed by incubation with compound treat-ment for an additional 48 hours. Viral RNA was evaluated by quantitativereal-time polymerase chain reaction (qRT-PCR). (B) Primary human hepato-cytes were infected with wild-type HCVcc in the presence of (S)-CCZ titra-tion overnight followed by incubation with (S)-CCZ titration for anadditional 48 hours. Intracellular viral RNA levels were evaluated by qRT-PCR. (C) In the presence of compound treatment, Huh7.5.1 cells were pas-saged every 3 days for seven passages and were plated on 96-well plates3 days before ATPlite assay to measure cell viability. (D) HCV replicationcycle assays were carried out with (S)-CCZ at 10 mM. Cyclosporin A (10 mM)was a control in HCV single-cycle infection (HCVsc), transient repliconwww.Sc
(10 nM) was used as a control in HCV pseudoparticle (HCVpp) genotype1a and 1b, vesicular stomatitis virus G pseudoparticle (VSV-Gpp), and murineleukemia virus pseudoparticle (MLVpp) assays. Results were normalized toDMSO. RLU, relative luminescence units. GT, genotype. (E) At t = −2 hours,HCV-Luc was incubated with Huh7.5.1 cells at 4°C for 2 hours for attach-ment. At t = 0 hours, the unbound virus was removed, and the plates weremoved to 37°C to allow synchronous infection and incubated for 48 hoursbefore virus load measurement. (S)-CCZ (10 mM), bafilomycin A1 (10 nM), andsofosbuvir (10 mM) were added either continuously or at the indicated timepoints and incubated for 2 hours. (F) Results from (E) were normalized toDMSO continuous treatment. Data are means of replicates ± SEM (n ≥ 3).*P < 0.05, **P < 0.005, ***P < 0.0001, versus DMSO (Student’s t test).
Table 1. Anti-HCV activity, selectivity, and anti-histamine propertiesof CCZ analogs. Compounds were tested in the HCV-Luc infection assayin parallel with the ATPlite assay. HCV-Luc was used to infect Huh7.5.1 cellsin the presence of compound titration. Viral infection and replication weremeasured by luciferase signal 48 hours after treatment, and cytotoxicitywas evaluated by the adenosine 5′-triphosphate (ATP)-based cell viability
assay. The concentration values that led to 50% and 90% viral inhibition(EC50 and EC90, respectively) and 50% cytotoxicity (CC50) were calculatedwith GraphPad Prism using a nonlinear regression equation. Cyclosporin Awas a control. Data are means ± SEM from n ≥ 3 independent experiments.Antihistamine activity was obtained with the b-arrestin H1-histamine recep-tor assay. N.D., not determined.
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Antihistamine activity at 10 nM (%)
Racemic CCZ
0.044 ± 0.011 1.40 ± 0.45 49.8 ± 17.2 994 72.60(R)-CCZ
0.020 ± 0.005 1.09 ± 0.37 37.5 ± 4.15 1875 88.00(S)-CCZ
0.024 ± 0.009 1.44 ± 0.43 33.4 ± 2.44 1392 41.70(S)-Nor-CCZ
0.034 ± 0.012 0.578 ± 0.099 9.31 ± 0.04 274 2.24Cyclosporin A
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genotype viruses (1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a, and 7a), the extracellularHCV RNA levels and infectivity [median tissue culture infectious doseper milliliter (TCID50/ml)] were inhibited by (S)-CCZ at 10 mM, sug-gesting pan-genotypic activity (table S3). Furthermore, (S)-CCZ showedno cytotoxicity at 1.0 and 5.0 mM and low cytotoxicity at 10 mM inHuh7.5.1 cells after continuous treatment for 21 days (Fig. 2C).
Mode of action of CCZ on HCV replication cycleTo better understand what stage of the viral replication cycle CCZanalogs target, we performed HCV single-cycle infection assay,HCV subgenomic replicon assays, and HCVpp assays in human hepa-tocytes with (S)-CCZ. For the HCV single-cycle infection assay, the core-defective, single-round infectious HCV genome (HCVsc, genotype 2a)can infect and replicate in hepatocytes but does not assemble intonew virions. Thus, this assay assesses compounds with an effect onthe HCV replication cycle stages before virion assembly. (S)-CCZ exhib-ited strong inhibitory activity in the HCVsc assay, suggesting that CCZanalogs inhibit the early stage of HCV infection (Fig. 2D).
We further performed the HCV subgenomic replicon assays toevaluate whether these compounds would target viral RNA replica-tion. (S)-CCZ was used to treat genotype 1b and 2a HCV replicon celllines (12) and did not exert a significant inhibitory effect (Fig. 2D).HCV replication was not inhibited with (S)-CCZ treatment in Huh7.5.1cells transiently transfected with a genotype 1a replicon (Fig. 2D).These results indicate that CCZ analogs do not target viral RNA repli-cation. (S)-CCZ also demonstrated no significant inhibitory effect onHCVpp (defective retroviral particles that harbor HCV envelope gly-coproteins) entry into Huh7.5.1 cells with VSV-Gpp and MLVpp ascontrol pseudoviruses (Fig. 2D).
The inhibitory kinetics of CCZ analogs on HCVcc infection wasaddressed in the time-of-addition experiment with (S)-CCZ, theHCV entry inhibitor bafilomycin A1, and the replication inhibitor so-fosbuvir (Fig. 2E). (S)-CCZ exhibited potent inhibition when addedduring viral attachment at 4°C for 2 hours or during the first 2 hoursof synchronous entry at 37°C, and both effects were comparable to thatof continuous (S)-CCZ treatment (Fig. 2F). Less inhibitory effect wasobserved when (S)-CCZ was added 1 hour after synchronous entry,and no significant inhibition was detected when it was added 2 or 3 hoursafter entry. Bafilomycin A1, reported to modulate post-entry fusion (13),only showed significant inhibitory effect when added during the first 2hours of synchronous entry (Fig. 2, E and F). Sofosbuvir, an RNA re-plication inhibitor targeting NS5B polymerase (14), inhibited HCVccinfection when added at all the time points except during the attachmentstage (Fig. 2, E and F). The comparison of the kinetics of (S)-CCZ withthat of bafilomycin A1 and sofosbuvir suggests that CCZ analogs inhibitviral infection possibly at a late-entry step before RNA replication.
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When evaluated in an immunofluorescence assay and/or Westernblotting, (S)-CCZ treatment did not alter the protein level or cellulardistribution of known HCV entry factors, including CD81, claudin-1,occludin, Niemann-Pick C1-like 1 (NPC1L1), and scavenger receptorclass B1, suggesting that the anti-HCV mechanism of (S)-CCZ is notdirectly related to affecting the expression levels or cellular distributionof these entry factors (fig. S1). Further investigation is needed toelucidate its mode of action.
Synergistic antiviral effect of CCZ in combination withanti-HCV drugsIn the treatment of chronic HCV infection, combination regimenslower the chance of developing drug-resistant viral mutations. We eval-uated the anti-HCV activity of (S)-CCZ in combination with differentclasses of anti-HCV drugs and agents at various concentrations. Thecombination of (S)-CCZ and each drug led to a greater HCV inhib-itory effect than either of them alone in a dose-dependent manner,without cytotoxicity (fig. S2). Using the MacSynergy II program basedon the Bliss independence model (15), we generated three-dimensionalsurface plots (fig. S2) and calculated the log volume of synergism foreach combination (Table 2). The results were also analyzed with theCalcuSyn program (16) in which the combination indices were calcu-lated (Table 2). The antiviral effect of (S)-CCZ was highly synergisticwith ribavirin, IFN-a, telaprevir, boceprevir, sofosbuvir, daclatasvir, andcyclosporin A, without significant cytotoxicity, supporting its potentialusage in combination therapy with these drugs.
Lack of antiviral effect of CCZ against multiple types of virusesBoth HCV and dengue virus are members of the Flaviviridae family.(S)-CCZ demonstrated an EC50 value of 1.88 mM in the dengue reportervirus particle (RVP) assay, which is more than 70-fold higher than itsEC50 value on HCV-Luc infection (fig. S3 and Table 1). The CC50 value(>31.6 mM) in this assay was consistent with the previous observationwhen cells were infected with HCV-Luc (fig. S3 and Table 1). Lycorine-HCl was tested as a positive control (EC50 = 0.0406 mM; CC50 > 31.6 mM).
Although CCZ has reported activity against HIV (17), (S)-CCZ hadlittle or no antiviral activity (SI < 10 and/or EC50 > 2 mM) in the Na-tional Institute of Allergy and Infectious Diseases (NIAID) antiviralscreen against 13 types of viruses: hepatitis B virus, HCV replicon,herpes simplex virus-1, human cytomegalovirus, vaccinia virus, denguevirus, influenza A (H1N1) virus, respiratory syncytial virus, SARS (se-vere acute respiratory syndrome) coronavirus, poliovirus 3, Rift Valleyfever virus, Tacaribe virus, and Venezuelan equine encephalitis virus(table S4). It is worth noting that the NIAID panel used an infectiousdengue virus in the plaque-forming assay to detect anti-dengue virusactivity and therefore is more reliable.
Table 2. Synergistic antiviral effect of CCZ in combination with anti-HCV drugs. The level of synergy was defined in MacSynergy as follows:++, moderate synergy (5 ≤ log volume < 9); +++, strong synergy (logvolume ≥ 9). Combination indices (CI) are means ± SEM from combi-
nations of the tested drug with (S)-CCZ at or near their EC50 valueswhen tested alone (n ≥ 6). The level of synergy was defined by Calcu-Syn as follows: ++, moderate synergy (0.7 ≤ CI < 0.85); +++, synergy(0.3 ≤ CI < 0.7).
Program
Parameter Ribavirin IFN-a TelaprevirienceTra
Boceprevir
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Cyclosporin A
MacSynergy
Log volume +++ +++ +++ +++ +++ +++ ++CalcuSyn
CI value 0.630 ± 0.106 0.609 ± 0.128 0.426 ± 0.138 0.691 ± 0.114 0.362 ± 0.075 0.427 ± 0.142 0.727 ± 0.187Synergy volume
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In vitro and in vivo ADME and pharmacokineticproperties of CCZBecause CCZ was developed as a first-generation antihistamine morethan 50 years ago, many of its pharmacologic properties were notavailable. To determine its ADME (absorption, distribution, metabo-lism, and excretion) and pharmacokinetic properties, we evaluatedCCZ and nor-CCZ in the microsomal stability assay with human andmouse microsomes. CCZ and nor-CCZ both exhibited long half-lives(T1/2) when incubated with human microsomes (T1/2 > 100 min)(Fig. 3A). However, the intrinsic clearance of CCZ was faster than thatof nor-CCZ. Nor-CCZ showed more than fourfold longer T1/2 in micethan that of CCZ.
In vitro activity in the presence of 40% human serum in conjunc-tion with pharmacokinetics have been used to predict the inhibitoryquotient (IQ) value of antimicrobial agents, namely, the ratio of com-pound exposure to microbial susceptibility. The protein binding
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adjusted EC50 value of (S)-CCZ in 40% human serum was 0.240 mM,which was an 8.7-fold increase relative to that in 10% fetal bovineserum (FBS), suggesting some potential for plasma protein binding(Fig. 3B).
The pharmacokinetic properties and tissue distribution of (S)-CCZwere evaluated in mice with a single intraperitoneal dose at 50 mg/kg.Depending on dosing three or two times per day (t.i.d. or b.i.d, respec-tively), the plasma concentration of (S)-CCZ at 8 or 12 hours afteradministration (2.02 or 0.592 mM, respectively) could be considered asCmin (Fig. 3C). From Cmin and the protein binding adjusted EC50 value,the IQ values were calculated: if dosing t.i.d., IQ = Cmin/serum-adjustedEC50 value = 8.42; if dosing b.i.d., IQ = 2.47.
Because HCV infection and replication occur only in hepatocytes,it is essential for anti-HCV compounds to achieve high liver exposure.(S)-CCZ showed a significantly higher exposure in the liver than in theplasma at all time points (Fig. 3, C andD). The average concentration in the
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liver during 0 to 24 hours was 195 mmol/kg,whereas the average plasma concentrationwas 6.58 mM, resulting in a ratio of liverto plasma of 29.6 to 1 (assuming the den-sity of the liver = 1.0 g/ml). It is knownthat CCZ can cross the blood-brain bar-rier and lead to side effects, such as seda-tion. Thus, the brain level of (S)-CCZ wasalso measured (Figure 3, C and D). Brainlevels were comparable to liver levels forboth (S)-CCZ and (S)-nor-CCZ, suggestingcentral nervous system (CNS) penetrationof the drugs and potential side effects thatwould need to be taken into considerationfor future development.
(S)-Nor-CCZ, the primary metaboliteof (S)-CCZ, was also tested for pharmaco-kinetics and tissue distribution at 10 mg/kgthrough the intraperitoneal route. Com-pared to (S)-CCZ, it showed a longer T1/2and higher liver distribution (ratio of av-erage concentration in liver to plasma of60.7 to 1) (Fig. 3, C and D). The plasmaconcentration of (S)-nor-CCZ at 8 and12 hours after administration was 0.890 and0.561 mM, respectively. Although (S)-nor-CCZ was dosed at 10 mg/kg, the plasmalevel at 12 hours was comparable withthat of (S)-CCZ dosed at 50 mg/kg. Over-all, (S)-nor-CCZ showed preferable phar-macokinetic properties to (S)-CCZ in mice.These results are consistent with the re-sults obtained in the mouse microsomalstability assay (Fig. 3A).
CCZ inhibits HCV infectionin vivo without evidence ofdrug resistance(S)-CCZwas tested in an albumin–urokinaseplasminogen activator/severe combined im-munodeficient (Alb-uPA/SCID) chimericmouse model infected with HCV genotype
Fig. 3. In vitro and in vivo ADME and pharmacokinetics of CCZ. (A) The microsomal stability of CCZand nor-CCZ was measured in vitro by incubation with human or mouse microsomes. Intrinsic clearance
(Clint) and T1/2 were calculated. (B) The protein binding adjusted EC50 and CC50 values of (S)-CCZ weremeasured in 40% of human serum (HS) with HCV-Luc and ATPlite assays. (C and D) Plasma, brain, and liverconcentrations of the drug were measured over time after a single intraperitoneal dose of (S)-CCZ (50 mg/kg)or (S)-nor-CCZ (10 mg/kg). Results are means of replicates ± SEM (n = 3). Asterisks indicate statistical sig-nificance of liver concentration compared with plasma concentration by Student’s t test (*P < 0.05, **P <0.005, ***P < 0.0001) (C). T1/2, the highest concentration after administration of compounds (Cmax), andtime to reach Cmax (Tmax) are provided in (D).8 April 2015 Vol 7 Issue 282 282ra49 5
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1b or 2a. On the basis of the pharmacokinetic data above, doses of50 and 10 mg/kg daily (for 4 weeks in genotype 1b infection and for6 weeks in genotype 2a infection) were used and led to a time-dependent reduction of HCV titers from the pretreatment baselinesin mice infected with HCV (2 log in genotype 1b and 1.5 log in geno-type 2a, respectively) (Fig. 4A). Doses as low as 2 mg/kg daily for4 weeks also caused a decrease of genotype 1b virus titer (about 1 log).A rebound of virus titer after stopping treatment was observed in bothgenotype infections. However, HCV titers continued to decline duringthe treatment period without rebound, suggesting the absence ofemergence of drug-resistant virus.
Nor-CCZ was found to be similarly active in vitro against HCVinfection as CCZ but demonstrated a preferable pharmacokineticprofile in the mouse model. It is possible that part of the antiviral ac-tivity observed on (S)-CCZ in vivo was due to the conversion to (S)-nor-CCZ. To detect the metabolism of (S)-CCZ to (S)-nor-CCZ in an
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Alb-uPA/SCID chimeric mouse model and to confirm the pharmaco-kinetic properties and tissue distribution of these two analogs, we eval-uated the concentrations of (S)-CCZ and (S)-nor-CCZ in serum, liver,and brain samples from these chimeric mice during the in vivo effi-cacy experiment. There was no significant difference in the concen-tration of (S)-CCZ in mice infected with HCV of different genotypes(n ≥ 14; P > 0.5, two-sided Student’s t test), and a slightly higher con-centration of (S)-nor-CCZ was observed in the genotype 1b group(Fig. 4B).
(S)-Nor-CCZ was detected in plasma from all dosing conditionswith about 5- to 10-fold higher concentration than that of (S)-CCZ.After 28 days of treatment, (S)-nor-CCZ was more concentratedthan (S)-CCZ in all tissues (Fig. 4C). The liver to plasma ratios of(S)-CCZ were 41 and 38 (in the 10 and 2 mg/kg groups, respective-ly), which is consistent with what has been observed in the CD-1 phar-macokinetic mouse model (Fig.3, C and D). At a dose of 10 mg/kg,
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the liver concentrations of (S)-CCZ and(S)-nor-CCZ (2.66 and 12.1 mM respec-tively) were higher than their in vitroEC90 values (1.44 and 0.578 mM, respec-tively). At a dose of 2 mg/kg, although theliver concentration of (S)-CCZ (0.342 mM)was below its EC90 value, the (S)-nor-CCZlevel (3.16 mM) was still above its EC90
value (Fig. 1C), which might contributeto the observed minor decrease of HCVtiters.
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DISCUSSIONDespite the recent approval of direct-acting antivirals and multiple drug candi-dates in the pipeline for the treatment ofHCV infection, an affordable yet effica-cious treatment for this viral infection isan unmet clinical need. The repurposingor repositioning of existing drugs for dis-eases other than what the drugs were ap-proved for may streamline and facilitatepharmaceutical development (7). It is wellknown that polypharmacology is com-mon for most drugs and often explainstheir side effect profiles in human appli-cation. Many studies have reported drugsfor other purposes having antiviral activ-ity against various viral infections, butnone of them have proceeded to potentialclinical application (18–20). To this end,we performed a qHTS of approved drugsin the NPC, with the aim of identifyingnew antivirals for HCV treatment. Here,we described the discovery and preclinicalcharacterization of CCZ as an anti-HCVagent. Its potent anti-HCV activity bothin vitro and in vivo and high liver distribu-tion makes CCZ a promising candidatefor the treatment of HCV infection.
Fig. 4. In vivo efficacy and pharmacokinetics of CCZ in mice infected with HCV genotype 1b or 2a.Alb-uPA/SCID mice were engrafted with primary human hepatocytes and then infected with HCV serum
samples of genotype 1b or 2a. The mice were monitored for serum HCV RNA and human albumin for 4 to6 weeks before treatment. Pretreatment HCV RNA values were determined by averaging HCV RNA levels atweeks −2, −1, and 0. (A) Changes in the genotype 1b and 2a HCV titers from pretreatment baseline overtime. For 1b, over a period of 4 weeks during (S)-CCZ treatment and 4 weeks of follow-up with or withouttreatment. For 2a, over a period of 6 weeks of (S)-CCZ treatment and 4 weeks of follow-up without treatmentin both groups. The result at each last dosing time point (4 and 6 weeks for 1b and 2a, respectively) wascompared to the corresponding pretreatment level using the Mann-Whitney test after the Shapiro-Wilk nor-mality test. Significant difference in HCV RNA was obtained with all the treatment conditions (P < 0.05). (B)The concentrations of (S)-CCZ and (S)-nor-CCZ were measured in plasma samples collected at weeks 1, 2, 3,and 4 in genotype 1b–infected mice and at weeks 1, 2, 3, 4, 5, and 6 in genotype 2a–infected animals. Theweekly concentrations were averaged for each dosing group and shown. (C) The concentrations of (S)-CCZand (S)-nor-CCZ in plasma, liver, and brain samples collected at day 28 in genotype 1b–infected mice weremeasured by liquid chromatography–mass spectrometry (LC-MS). Data are means of mice in each group ±SEM (for genotype 1b, n = 5 in the 50 mg/kg group, n = 4 in the 10 mg/kg group, and n = 5 in the 2 mg/kggroup; for genotype 2a, n = 8 in the 50 mg/kg group and n = 5 in the 10 mg/kg group).8 April 2015 Vol 7 Issue 282 282ra49 6
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Cyclizine and phenothiazine were the two most potent series of H1-antihistamines with anti-HCV activity that we identified from theqHTS of the NPC (table S1). One of the antihistamines, clemizole,was shown previously to have anti-HCV activity by interfering withthe nonstructural protein 4B functions (21), but our screen indicated amuch less potent activity of clemizole as compared to CCZ. The anti-HCV properties of cyclizines and phenothiazines have been reportedpreviously and are consistent with our results (22–26). Compared tothe phenothiazines in table S1, CCZ analogs were chosen for fur-ther studies on the basis of the following aspects: (i) higher potencyand selectivity against HCV than phenothiazines, (ii) fewer CNS-related side effects than phenothiazines, and (iii) potential for furtherstructure modification to reduce side effects and increase anti-HCVactivity.
Cyclizine H1-antihistamines bind to the H1-histamine receptor andinhibit the binding of histamine as inverse agonists (27). (R)-CCZ ismore active as an antihistamine than (S)-CCZ, whereas both enantio-mers are equally active in inhibiting HCV infection. Nor-CCZ, whichis a metabolite of CCZ but has little antihistamine activity, has a simi-larly potent anti-HCV activity. Histamine also has no effect on HCVinfection (table S1). Therefore, the anti-HCV activity of CCZ analogsis unlikely to be attributed to their action on the H1-histamine receptor.
Instead, CCZ may act through a novel mode of action by inhibitinglate-stage HCV entry but not affecting viral replication or assembly/secretion. CCZ exhibited no inhibitory effect on the entry of HCVpp.There is growing evidence of HCV entry inhibitors with no activityagainst HCVpp, such as the NPC1L1 antagonist ezetimibe and humanapolipoprotein E peptides (13, 28). Therefore, the lack of an effect inthe HCVpp system by an agent does not necessarily exclude entry as atarget. The agent could be targeting an entry step of HCV infectionthat is not otherwise captured by the HCVpp system. When HCVccwas used to evaluate its inhibitory kinetics, the inhibitory effect of (S)-CCZ on viral level during the 2-hour viral attachment and the first2 hours of synchronous entry was comparable to that of continuous(S)-CCZ treatment for 48 hours, although no significant inhibitionwas detected when (S)-CCZ was added 2 or 3 hours after entry. Con-versely, treatment with bafilomycin A1 and sofosbuvir during theattachment step had no effect on viral level. The sustained activityof CCZ when incubated during the attachment step could be ex-plained by a longer off rate from its target, whereas bafilomycinmay have a rapid off rate and the sofosbuvir’s target has not beenformed yet during the viral attachment step. Overall, these data sug-gest that (S)-CCZ inhibits HCV late-entry step before RNA replica-tion. On the basis of the kinetics and other virologic assays, (S)-CCZprobably functions at a step somewhat later than where bafilomycinA1 acts to block viral fusion, suggesting a novel target of CCZ in HCVinfection that needs to be explored further. An HCVcc “fusion” as-say has been developed previously, but the assay does not measurefusion directly because inhibitors of any of the viral entry steps beforefusion would score positive in this assay (28).
Various drugs and anti-HCV agents with distinct mechanisms ofaction were tested in combination with (S)-CCZ against HCV infec-tion in vitro. The mechanism of action of ribavirin and IFN-a is me-diated through host antiviral response. Telaprevir and boceprevir areNS3/4A serine protease inhibitors, daclatasvir inhibits HCV NS5A,and sofosbuvir is an NS5B polymerase inhibitor (2, 14, 29). CyclosporinA targets viral RNA replication (30). The strong synergism of (S)-CCZwith all these agents not only supports its use in combination therapy
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but also indicates that (S)-CCZ inhibits HCV infection through a dif-ferent mechanism, such as targeting viral entry.
(S)-Nor-CCZ demonstrated better pharmacokinetic propertiesthan those of (S)-CCZ, namely, a longer T1/2 and higher liver distribu-tion. Consistent results were observed in the mouse microsomal sta-bility assay and pharmacokinetic studies during the efficacy evaluationin HCV-infected chimeric mice. These observations in mice maynot reflect the ADME in humans. Indeed, CCZ and nor-CCZ showedless of a difference in T1/2 in the human microsomal stability assay. Onthe other hand, nor-CCZ was less selective in vitro than CCZ (SI =188 and 758, respectively). Overall, CCZ is preferable for further de-velopment, particularly because its safety profile is established inhumans. One potential drawback of using CCZ to treat HCV infectionis its brain distribution—the level of which is comparable to its liverdistribution in mouse pharmacokinetic studies. Reducing blood-brainbarrier penetration would thus be important for the future develop-ment of this class of drugs.
Entry inhibitors typically should not affect established infectionwhen used alone in vivo. However, the in vivo antiviral effect ofCCZ on preexisting steady-state infection in mice is similar to thatof IFN-a (31). It is known that in vivo, circulating HCV has a veryshort T1/2 (32), HCV-infected hepatocytes may turnover quickly, andthe newly produced virus probably continues to infect uninfected he-patocytes (33). If viral reinfection and spread can be effectively pre-vented in the infected liver, a reduction in viremia can be achieved.In addition, recent data suggested that cell-to-cell spread may be animportant pathway for viral dissemination in vivo (34). It is possiblethat CCZ might be particularly effective in blocking this pathway. Fi-nally, the kinetics of decline in viremia in CCZ-treated mice is a steadyand linear decline, which is quite different from that of direct-actingantivirals, which typically have a rapid first and then a slow second-phase decline. Studies have shown similar reduction of HCV titer inclinical trials as that of the efficacy study using the SCID/uPA mousemodel, suggesting the applicability of this mouse model in predictingantiviral activity in humans (35).
A dose response of anti-HCV activities against both genotype1 and 2 by CCZ treatment was observed in the SCID/uPA mousemodel. The anti-HCV activity of CCZ was initially discovered usingthe infectious HCV genotype 2a system; however, (S)-CCZ showedeven higher efficacy on genotype 1b infection in the mouse model.This finding suggests that (S)-CCZ may have pan-genotypic in vivoactivity, which is consistent with the pan-genotypic in vitro activityshown in table S1.
Several FDA-approved drugs have shown anti-HCV activities andcan potentially be repurposed for the clinical treatment of HCV, suchas erlotinib and dasatinib (anticancer drugs), ezetimibe (cholesteroldrug), and ferroquine (antimalarial) (28, 36, 37). Here, CCZ showedmore compelling in vitro and in vivo activity against HCV infection thanthese drugs. Although in vitro activity cannot adequately be comparedbetween different assays with different strains or reported virus, it isworth noting that the above drugs do not have in vitro EC50 values com-parable to that of CCZ (~50 nM). Some of the drugs, like erlotinib anddasatinib, also have substantial side effects. CCZ is also advantageousbecause of its established safety profile in patients for a long period oftime (25 mg per tablet by mouth every 6 to 8 hours, not to exceed threetablets in 24 hours) and cost-effectiveness ($0.55 per tablet).
In summary, this study provides compelling evidence that CCZ, anover-the-counter allergy drug, has a strong anti-HCV activity in vitro
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and in vivo in a mouse model of HCV infection. On the basis of theseresults, a clinical assessment of CCZ alone and in combination withother anti-HCV drugs is warranted. The repurposing or repositioningof CCZ in HCV treatment may provide a more affordable alternativeto the current costly options, especially in low-resource settings wherechronic HCV infection is endemic. This study also lays the foundationfor further structure modification of CCZ to discover more optimalanalogs for the treatment of HCV infection.
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MATERIALS AND METHODS
Study designThe objective of this study was to identify anti-HCV compounds fromexisting, FDA-approved drugs through qHTS of the NPC library (9)and to repurpose candidate drugs for the treatment of HCV infection.The identified drugs with potent anti-HCV activity were characterizedin vitro, with a focus on confirming antiviral properties, target iden-tification in the HCV replication cycle, and activity in combinationwith other clinically used anti-HCV drugs. In vivo studies were carriedout to evaluate candidate drug pharmacokinetic properties and effica-cy in an Alb-uPA/SCID mouse model, which has been successfullyused for translational drug development, including for HCV infection(35, 38). This study conducted controlled laboratory experiments withcell culture and animals. The animal study protocols were approvedby the Institutional Animal Care and Use Committee (IACUC) of NIHfor the mouse pharmacokinetic study and by the IACUC of HiroshimaUniversity, Japan, for the mouse efficacy study. Power analysis was notperformed, and replication conditions for each experiment are definedand described in the figure legends. The animals were randomized,but the investigators were not blinded to the experimental conditions.
Primary qHTS and secondary confirmation assaysHuman hepatocytes and viruses were obtained and maintained as de-scribed in Supplementary Materials and Methods. The suppliers of thecompounds in the NPC were published previously (9). In the primaryscreen using the cell-based HCV infection assay, the qHTS was per-formed as described previously using a fully automated roboticscreening system (8). Dose-response curves and EC50 and CC50 valueswere generated through a seven-concentration titration for each com-pound from the NPC. Curve classes were defined on the basis of thequality of the curve fitting derived from the Hill equation (39). Primaryhits were selected with the dose-response curves in class 1 or 2 in theanti-HCV luciferase assay and class 3 or 4 in the ATPlite assay (cyto-toxicity). A two-part HCV infection assay, as described before, wascarried out to confirm the primary hits.
HCVcc infection assayHuh7.5.1 cells were seeded in 12-well plates (1 × 105 cells per well)and cultured overnight. Wild-type HCV genotype 2a and chimericgenotype 1a, 1b, 2b, 3a, 4a, 5a, 6a, or 7a was used to infect the cellswith the treatment of compounds. Virus-containing medium was re-moved after overnight incubation, and compound treatment wasadded back followed by incubation for an additional 48 hours. Cyclo-sporin A (Sigma-Aldrich) was used as a positive control. Intracellularand extracellular viral RNA levels were evaluated by qRT-PCR. Thesame methods were used with primary human hepatocytes in 24-wellplates. For TCID50/ml determination, medium collected was used to
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infect naïve Huh7.5.1 cells attached in 96-well plates in serial dilutions.TCID50/ml was calculated as described on the basis of the dilution atwhich 50% of the wells were positive for HCV (40).
In vitro combination evaluationHuh7.5.1 cells were seeded in 96-well plates (1 × 104 cells per well) andcultured overnight. In the presence of (S)-CCZ and the agent of interesttitrated in vertical and horizontal, respectively, HCV-Luc infection assaywas carried out in parallel with ATPlite assay. The anti-HCV effect of(S)-CCZ in combination with each agent was analyzed using two inde-pendent mathematical models, the Bliss independence model and theLoewe additivity model (15, 16), to predict the theoretical additive, syn-ergistic, or antagonistic effect (Supplementary Materials and Methods).
ADME and pharmacokineticsIn vitro pharmacokinetic studies are described in SupplementaryMaterials and Methods. The in vivo pharmacokinetic properties ofthe compounds were measured in the plasma, brain, and liver of maleCD-1 mice after a single intraperitoneal administration. The plasmaand tissue concentrations of the compounds at various time points upto 24 hours after administration were determined by LC-MS analysis.
In vivo efficacy studies in chimeric mouse modelAlb-uPA/SCID mice were engrafted with primary human hepatocytesand then infected with HCV serum samples of genotype 1b or 2a. Themice were monitored for serum HCV RNA and human albumin for4 to 6 weeks before treatment. Genotype 1b HCV titers were moni-tored in HCV-infected chimeric mice over a 4-week period of (S)-CCZ treatment. A 4-week follow-up monitoring without treatmentwas carried out only in the group that received the 50 mg/kg dose.The means of changes in serum HCV RNA level in each group werecalculated (n = 5 in the 50 mg/kg daily group, n = 4 in the 10 mg/kgdaily group, and n = 5 in the 2 mg/kg daily group). Genotype 2a HCVtiters were monitored over a period of 10 weeks with 6 weeks of (S)-CCZ treatment and 4 weeks of follow-up without treatment in bothgroups in HCV-infected chimeric mice. The means of changes inserum HCV RNA level in each group were calculated (n = 8 in the50 mg/kg daily group and n = 5 in the 10 mg/kg daily group). Humanserum albumin was measured in parallel for control.
Statistical analysisStatistical significance was assessed with GraphPad Prism 5.0 software.Data are presented as means ± SEM (n ≥ 3). Student’s t test was usedto determine whether the means of two groups are significantly differ-ent on the basis of one continuous variable when normal distributionwas assumed in a small sample size. Normal distribution was exam-ined by the Shapiro-Wilk normality test. In case of nonparametricdistribution, the Mann-Whitney test was used. In all analyses, two-sidedP values were used, and P < 0.05 was considered statistically significant.
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/7/282/282ra49/DC1Materials and MethodsFig. S1. CCZ does not affect the expression levels or cellular distribution of HCV entry factors.Fig. S2. Synergistic antiviral effects of CCZ in combination with anti-HCV drugs.Fig. S3. Antiviral activity of CCZ against dengue virus.Table S1. Structure, anti-HCV activity, and cytotoxicity of H1-antihistamine compounds fromthe NPC library.
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Table S2. Negligible amount of transformation of (S)-CCZ to (S)-nor-CCZ in vitro.Table S3. Antiviral activity of CCZ against HCV genotypes 1 to 7.Table S4. NIAID antiviral screen of CCZ against 13 viruses.References (41–49)
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Acknowledgments: We thank B. Zhang for technical assistance; S. Michael and M. Balcom forassistance in robotic control in qHTS; and P. Shinn, M. Itkin, and D. van Leer for help withcompound management. Funding: The Intramural Research Program of the National Instituteof Diabetes and Digestive and Kidney Diseases and Molecular Libraries funding. Author con-tributions: S.H., N.S., M.F., W.Z., K.C., J.J.M., and T.J.L. designed the research; Z.H. performed theqHTS and secondary confirmation assays for the screening of compounds; X.H. and N.S. ana-lyzed qHTS and secondary confirmation assay data; S.H., B.L., and Z.H. performed the HCV-Lucinfection, ATPlite, and wild-type HCVcc infection assays; S.H. performed the H1-histamine re-ceptor assay, HCV replication cycle assays, and time-of-addition assay; Z.H. performed thelong-term cytotoxicity assay; C.J.S. performed the immunofluorescence assays and Westernblotting; S.H., B.L., and V.C. performed the in vitro combination evaluation. S.H., B.L., V.C.,Z.H., C. J. S., and Q. L. analyzed in vitro assay data; J.X. performed chemical isolation; A.Q.W.handled animals and performed pharmacokinetic studies, and A.Q.W. and S.H. analyzed related
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data; M.I., N.H., and K.C. handled animals and performed animal efficacy studies, and S. H.analyzed related data; X.H., N.S., and S.H. performed statistical analysis; S.H., B.L., V.C., Z.H.,X.H., J.X., C.J.S., N.S., M.F., W.Z., J.J.M., and T.J.L. participated in the discussion of all data inperiodic collaboration meetings. S.H., Q.L., N.S., and T.J.L. wrote the paper. Competing interests:T.J.L., M.F., S.H., X.H., Z.H., J.J.M., J.X., and W.Z. are named as inventors on a patent related toantihistamines and heterocyclic compounds for the treatment of HCV. The other authors declarethat they have no competing interests. Data and materials availability: Primary human hepa-tocytes were provided by the NIH-funded Liver Tissue Procurement and Cell Distribution System(N01-DK-7-0004/HHSN26700700004C; principal investigator, S. Strom, University of Pittsburgh).The plasmids (pNL4-3.Luc.R-E- and pHEF-VSVG) were obtained from the NIH AIDS ReagentProgram (Division of AIDS, NIAID, NIH). pNL4-3.Luc.R-E- was originally from N. Landau.pHEF-VSVG was from L.-J. Chang. Plasmids encoding HCV genotype 1a envelope proteins wereprovided by S. Ray (Johns Hopkins University). HCV genotypes 1 to 7 were obtained from J. Bukh(Copenhagen University Hospital).
Submitted 7 August 2014Accepted 20 February 2015Published 8 April 201510.1126/scitranslmed.3010286
Citation: S. He, B. Lin, V. Chu, Z. Hu, X. Hu, J. Xiao, A. Q. Wang, C. J. Schweitzer, Q. Li,M. Imamura, N. Hiraga, N. Southall, M. Ferrer, W. Zheng, K. Chayama, J. J. Marugan,T. J. Liang, Repurposing of the antihistamine chlorcyclizine and related compounds for treatmentof hepatitis C virus infection. Sci. Transl. Med. 7, 282ra49 (2015).
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nceTranslationalMedicine.org 8 April 2015 Vol 7 Issue 282 282ra49 10
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hepatitis C virus infectionRepurposing of the antihistamine chlorcyclizine and related compounds for treatment of
T. Jake LiangLi, Michio Imamura, Nobuhiko Hiraga, Noel Southall, Marc Ferrer, Wei Zheng, Kazuaki Chayama, Juan J. Marugan and Shanshan He, Billy Lin, Virginia Chu, Zongyi Hu, Xin Hu, Jingbo Xiao, Amy Q. Wang, Cameron J. Schweitzer, Qisheng
DOI: 10.1126/scitranslmed.3010286, 282ra49282ra49.7Sci Transl Med
translation to HCV-endemic countries in Asia and Africa.. Antihistamines are widely available, safe, and inexpensive, making them ideal for imminentαA, and interferon-
hepatitis B, and showed synergy with different classes of anti-HCV drugs, such as ribavirin, sofosbuvir, cyclosporinantivirals. Chlorcyclizine was specific for HCV, demonstrating no activity against 13 other viruses, including
a common problem with existing−−and in mice with ''humanized'' livers, without evidence of drug resistance Among these, the first-generation antihistamine chlorcyclizine demonstrated high antiviral activity in cell culture . in a screen of a library of approved drugs, the NIH Chemical Genomics Center Pharmaceutical Collection.et al
cancer. The class of compounds, called antihistamines, which are used to relieve allergies, was uncovered by He a virus that often goes undetected, but can exacerbate many liver diseases, including cirrhosis and−−infection
A drug commonly used for a runny nose may now be repurposed for treating hepatitis C virus (HCV)Over-the-counter allergy drug inhibits viral infection
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